Preparation Methods for a Novel Generation of Biological Safe KLH Products Used for Cancer Treatment, for the Development of Conjugated Therapeutic Vaccines and as Challenging Agents

ABSTRACT

The present invention relates to the provision of a biologically safe hemolymph sera, preferably hemocyanin, more preferably KLH (keyhole limpet hemocyanin). The hemocyanin is purified using anion exchange chromatography.

BACKGROUND OF THE INVENTION

The present invention relates to the provision of a biologically safehemolymph sera, preferably hemocyanin, more preferably KLH (keyholelimpet hemocyanin).

Hemocyanin is a blue copper protein which occurs in a freely dissolvedform in the blood of numerous molluscs and arthropods and transportsoxygen. Of the molluscs, the cephalopods, chitons, most gastropods andsome bivalves contain hemocyanin. Among the arthropods, hemocyanin istypical of arachnids, xiphosurans, malacostracan crustaceans andScutigera. Numerous species of insects contain proteins which arederived from hemocyanin. Hemocyanins are present in the extracellularmedium and float in the hemolymph.

While arthropod hemocyanin has a maximum diameter of 25 nm under anelectron microscope and a subunit has a molecular weight of 75,000Dalton (Da), mollusc cyanins are much larger. Thus e.g. the hemocyaninof Megathura has a diameter of 35 nm and is composed of 2 subunits. Eachsubunit has a molecular weight of approx. 400,000 Da and is divided intoeight oxygen-binding domains, each of which has a molecular weight ofapprox. 50,000. The domains differ immunologically.

The hemocyanin of gastropods visible under an electron microscope has amolecular weight of approx. 8 million Da and is a didecamer. In contrastto this, the hemocyanin of cephalopods is arranged as an isolateddecamer, which also differs significantly from the hemocyanin ofgastropods in the quaternary structure.

Traditionally, hemocyanin was obtained from hemolymph from the Megathuracrenulata. More recently, the market for gastropod hemocyanins hasexpanded to include hemocyanin from Haliotis tuberculate and Concholepusconcholepus. The hemolymph from other gastropod molluscs is also underinvestigation for useful properties.

The hemocyanin of the Californian keyhole limpet Megathura crenulata isof particular immunological interest. The hemocyanin is therefore alsocalled keyhole limpet hemocyanin (KLH). Hemocyanins are very potentantigens. Immunization of a vertebrate leads to a non-specificactivation of the immune system which to date is not very wellunderstood. By the general activation of the immune system, it is thenpossible also to achieve an immune reaction to other foreign structureswhich have previously been tolerated. KLH is used above all as a haptencarrier in order thus to achieve the formation of antibodies against thehapten.

In addition to Megathura crenulata, the abalone Haliotis tuberculatealso belongs to the Archaegastropoda group, which is relatively old inrespect of evolution. It is known that Haliotis also produceshemocyanin.

Native KLH is found in the hemolymph (pH 6.0-8.0) in colloidal solutionas a didecamer (molecular weight: around 8 million Da) and asmultidecamers (molecular weight: 12 to about 32 million Da). Thequantitative distribution of these aggregates varies. The didecamers andmultidecamers of KLH are composed of 2 types of subunits with an averagemolecular weight of around 400,000 Da. The two different types ofsubunits as well as the two different aggregation types are due to thefact that native KLH is a mixture of two different types KLH 1 and KLH2.

KLH is a mixture of two different hemocyanins, which are called KLH1 andKLH2. The subunit of KLH1 is a 390 kDa polypeptide which consists ofeight globular domains called 1 a to 1 h according to their sequence inthe subunit. On the other hand, KLH2 has a molecular weight of 360 kDaand according to the most recent data also contains 8 domains, called 2a to 2 h. In vivo every type of subunit forms homo-oligomers, while nohetero-oligomers have been observed.

Hemocyanins may be obtained in farms from test animals. Methodsdescribed for collection of hemolymph involve inserting a needle into amuscle of the foot to penetrate the pedal blood sinus (Harris et al.,“Keyhole Limpet Haemocyanin: Negative Staining in the Presence ofTrehalose,” Micron, 26 (1): 25-33 (1995). Semi-automated systems wereestablished allowing the collection of high amounts of hemolymph withoutkilling the animals. The manufacturing procedures allow extractingcommercial quantities of hemocyanin from animals grown in a controlledenvironment (WO 02/085389, US2002/192633).

There are a variety of well-known methods for purifying hemocyanins fromcrude hemolymph, which is the biological source of hemocyanins. Thesemethods include differential centrifugation, gel-permeationchromatography, and ion-exchange chromatography (U.S. Pat. No.5,407,912). Purified hemocyanins are commercially available in manyforms.

The incorporation of hemocyanins into promising new therapeutic products(see e.g., Jurincic-Winkler et al., “Antibody Response to Keyhole LimpetHemocyanin (KLH) Treatment in Patients with Superficial BladderCarcinoma,” Anticancer Res., 16 (4A): 2105-10 (1996); and Biomira, Inc.Company Press Release, Biomira.com, 2001) has resulted in the need for asustainable supply of commercial quantities of hemocyanin produced underconditions that meet the health and safety standards imposed by theUnited States Food and Drug Administration and other regulatoryagencies.

BRIEF SUMMARY OF THE INVENTION

Due to their native origin, hemocyanins such as KLH, suffer from therisk of bio-contamination by pathogens such as pathogenic bloodingredients, such as toxins, bacteria, including endotoxins producedthereby, as well as viruses.

It is therefore an object of the present invention to provide means andways in order to minimize the bio-burden by pathogens in nativehemocyanin. This includes the further object of providing a process withwhich safe and highly pure hemocyanin can be prepared.

The present inventors have identified that blood and hemolymph takenfrom molluscs immediately harvested from the sea may be contaminated byviruses bacteria, toxins or endotoxins. There is, thus, a need forreducing the contamination in the molluscs and the blood or hemolymphobtained therefrom.

Conventionally, for the removal of viruses there are different processesthat can be used: virus inactivation, e.g. the treatment of the proteinusing the detergent/solvent method, ionised radiation, thermal treatment(ca. 60° C.) and the incubation at pH values<3. These processes cannotbe used in the treatment of hemocyanins such as KLH because they lead toa denaturisation of the protein. Due to the high molecular structure ofKLH it is extremely sensitive to these inactivating methods. Also otherapproaches such as virus nanofiltration that have been developed in thepast few years cannot be used. These filters do not allow a significantvirus removal rate due to the small size difference between viralcontamination and the target protein. Gelfiltration that works byseparating the molecular weight is also not suitable for the removal ofviral contaminants because the size of the viruses do not varysignificantly from those of hemocyanins.

Thus, there is a need to provide new means for reducing virus load inhemocyanins and for the separation of viruses from hemocyanins.

It is further an object underlying the present invention to providetherapeutically valuable amounts of hemocyanins, e.g. KLH, or subunitsthereof, such as immunocyanin. These amounts need to be provided in apurified and isolated manner. Accordingly, means and methods forisolating and purifying hemocyanins and subunits thereof (e.g. KLH orimmunocyanin) are needed.

The present inventors have tested several purification methods. Amongthese, ultracentrifugation is limited in handling due to limitedquantities of hemocyanin which be provided. It is further time consumingand accordingly expensive. There is a need for a cheaper isolation andpurification approach allowing the production of hemocyanin, such asKLH, or hemocyanin subunits, such as immunocyanin. Gelfiltrationchromatography was not successful. Hydrophobic interactionchromatography was selected for further evaluation but turned out to beunsuitable for the intended isolation and purification.

Ion exchange chromatography relies on charge-charge interactions betweenthe proteins in the sample and the charges immobilized on the concernedresin. Cation exchange chromatography employs positively chargedmolecules attached to a negatively charged solid support.

After conducting a laborious research program, the present inventorsfound that anion exchange chromatography, which employs negativelycharged molecules attached to a positively charged solid support, turnedout to be suitable for the intended isolation and purification. Morepreferably, a specific anion exchange column is provided comprising aspecific matrix material (see embodiment 1). In another preferredembodiment, a specific method for isolating and/or purifying hemocyanin,e.g. KLH, and/or its subunits, e.g. immunocyanin, is provided (seeembodiments 2-7).

Preferred embodiments of the invention are:

1. Anion exchange chromatography column comprising a matrix materialwith a particle size of more than 30 μm, preferably more than 40 μm, anda pore size of between 3 000 and 8 000 Å (Angstroem), preferably 6 000-8000 Angstroem.

2. A method of isolating and/or purifying hemocyanin or immunocyanincomprising the steps of:

-   -   a) providing a hemocyanin formulation derived from a marine        mollusc;    -   b) reducing the conductivity of the formulation provided in a)        to a value of between 10 mS/cm and 30 mS/cm by adding a dilution        buffer;    -   c) adding the diluted hemocyanin formulation obtained in step b)        to an anion exchange chromatography column;    -   d) rinsing the column with a buffer to purify the hemocyanin,        preferably to remove salts, and other proteins; and    -   e) eluting the hemocyanin or immunocyanin from the column by        adding a second buffer.

3. The method according to embodiment 2 wherein the buffer for purifyingis a buffer in the pH between 7 and 8.

4. The method of any of embodiments 2-3, wherein hemocyanin is eluted,wherein a buffer of a pH between 7 and 8 is used which includes Ca⁺⁺ andMg⁺⁺ ions.

5. The method according to any of embodiments 2-3, wherein immunocyaninis eluted, wherein a buffer is employed of a pH between 9 and 10 whichis free from Ca⁺⁺ and Mg⁺⁺ ions.

6. The method of any of embodiments 2-5, wherein the matrix material isan anion exchange matrix including particles of a particle size of morethan 30 μm, preferably more than 40 μm, more preferably 50 μm+/−5 μm.

7. The method of any of embodiments 2-6, wherein the anion exchangematrix includes pores of a pore size between 3 000 and 8 000 Angstroem,preferably 6 000-8 000 Angstroem.

8. Use of the anion exchange chromatography column of embodiment 1 forpurifying hemocyanin or its subunits.

In order to optimize the binding of charged hemocyanin or its subunits(KLH or immunocyanin) to the anion exchange column, the proteinformulation (solution or suspension) put onto the column needed to belowered in its conductivity. The selection of anion exchangechromatography among other isolation and purification methods as well asthe selection of a matrix material are described in Example 9.

One task the present inventors were faced with was the identification ofa most preferred matrix material. Due to the size of the hemocyanin andits subunits, a matrix material with small particles turned out not tobe useful. A particle size of at least 30 μm diameter turned out to behelpful. In a more preferred embodiment, the particle size is at least45 μm diameter, even more preferred are 50 μm+/−5 μm diameter. Thecapacity of the matrix material depended on the size of the“through-pores” or “pores”. A porous material with pores of at least3,000 Angstroem was found to be most preferred. The pore size may bebetween 3,000-8,000 Angstroem or preferably between 6,000-8,000Angstroem. The material may be a conventional material useful for ionexchange chromatography in the pH range of between 6-10, preferably inthe range between 7-9. Using anion exchange chromatography with such amatrix material achieved highest degrees of purity of more than 90%,preferably 95% or more preferably 99%. More than 15 mg/ml, preferablymore than 17 mg/ml, more preferably more than 23 mg/ml matrix materialof KLH are bound to the column.

Hemocyanin formulations such as hemolymph sera or subunits thereof, suchas immunocyanin, could not be applied directly to the column. Thepresent inventors identified that typical hemocyanin formulations, andas hemolymph sera derived from marine molluscs contain, apart fromhemocyanin and other serum components, high levels of sodium chlorideand other minerals. This leads to a very high conductivity in a range ofapproximately 50 mS/cm. Under these conditions, the protein formulation(e.g. the hemolymph serum) cannot be bound to the ion exchange column.In order to achieve a quantitative binding of the protein formulation, adilution in buffer was necessary. The buffer is a buffer in the pH rangebetween 6 and 8, preferably between 7 and 7.5. The buffer preferablyincludes Ca⁺⁺ and Mg⁺⁺ ions. The buffer needs to be suitable to dilutethe mineral and salt components. In particular, the buffer needs to besuitable to lower the conductivity. To achieve a quantitative binding ofthe hemocyanin formulation (or the protein formulation in more generalterms), the conductivity needs to be lowered below 25 mS/cm, preferablybelow 20 mS/cm. A preferred range is between 10 and 20 mS/cm.

A preferred medium is Poros® 50 HQ.

The linear flow rate of the sample may be between 10 ml/min and 100ml/min, preferably between 30 and 70 ml/min, most preferably 50 ml/min.

After reducing conductivity as described above, the protein formulation(the hemocyanin formulation) is loaded onto the anion exchange column.

Under neutral pH conditions (pH between 6 and 8, preferably between 7and 7.5), the hemocyanin binds to the anion exchange column. Elution ofother proteins, salts, minerals and/or other molecules is effected.Preferably, a buffer as used for diluting and reducing conductivity isemployed.

After the rinsing step, a purified protein from a hemocyanin formulationis bound to the anion exchange column.

If it is intended to elute the hemocyanin, such as KLH, withoutdissociation, the hemocyanin is eluted from the column using an elutionbuffer in the neutral range (pH between 6 and 8, preferably between 7and 7.5). The elution buffer is increased in its ionic strength ascompared to the rinsing buffer. In a preferred embodiment, the elutionbuffer comprises Ca⁺⁺ and Mg⁺⁺ ions, more preferably in a concentrationbetween 0.01 M and 0.1 M, even more preferably between 0.05 and 0.01 M,each. In a more preferred embodiment, the buffer alternatively oradditionally comprises TRIS/HCL, preferably in a concentration between0.1 and 1 M.

The difference between the rinsing and the elution buffer may be thesalt concentration.

Typically for elution, an elution gradient is applied employing 0.01 upto 1 M sodium chloride, more preferably between 0.05 and 0.7 M, mostpreferably between 0.075 and 0.575 M sodium chloride.

If it is intended to dissociate the hemocyanin into its subunits, e.g.KLH subunits or immunocyanin, before eluting, an alkaline buffer isadded (pH between 8 and 10, preferably between 9 and 10). Thedissociation buffer may be a dissociation buffer as described elsewherein the specification. Preferably, the dissociation buffer containsglycine and NaOH. It may also contain NaCl and/or EDTA. Under theseconditions, the hemocyanin dissociates into its subunits. Thedissociation buffer is free from Ca⁺⁺ and Mg⁺⁺ and is preferably used inan amount and concentration to remove Ca⁺⁺ and Mg⁺⁺ ions from thecolumn. The dissociation buffer can be used for elution as well. Again,an elution gradient with increased salt concentrations is employed foreluting. In the preferred embodiment, the elution gradient is asdescribed for hemocyanin as above.

To solve other problems, the present invention provides the methods andcompounds and compositions set forth below:

In a first aspect the present invention provides a method for thepreparation of hemolymph sera from a mollusc is provided, the methodcomprising a step of puncturing the pedal blood sinus of the molluscunder cold narcosis.

Preferably, the mollusc is Megathura crenulata. Other molluscs are e.g.Haliotis tuberculata (European Abalone), Haliotis rubra (AustralianAbalone)

In a preferred embodiment, during puncturing the mollusc is kept underspecific quarantine conditions, wherein the molluscs after beingobtained from their natural sources are kept in a quarantine aquariumsystem under conditions, wherein no organic feed is supplied and/or thewater in the aquarium system is purified by removing biologicalcontaminants. The removing of biological contaminants may includebio-filtration, protein skimming, etc.

The blood obtained upon puncturing may further be sterilized, preferablyby using 0.2 μm membrane filtration.

In a second aspect the present invention provides a method of isolatingnative hemocyanin comprising providing hemolymph sera, such as thoseobtained in the method of the first aspect, and isolating thehemocyanin, such as KLH, from hemolymph sera, preferably by employingdirect chromatography.

In a preferred embodiment, the direct chromatography is an ion exchangechromatography.

The method may further include a step of dissociating the hemocyanininto subunits of the hemocyanin oligomer. Optionally a step of purifyingthe hemocyanin subunits is performed as well. Optionally, a step ofre-associating the subunits into the oligomeric form of the hemocyaninis performed.

In a preferred embodiment the purification of hemocyanin subunits isperformed including a step of nanofiltration in order to removepotential biological contamination.

Preferably, the re-association step takes place comprising adiafiltration step or a dialysis step. The re-associated hemocyanin maybe finally purified by gel filtration.

Preferably, the hemocyanin is admixed stabilizing puffer system forlong-term storage.

In this aspect, also provided is a method of producing synthetichemocyanin such as synthetic KLH, comprising a step of dissociating thehemocyanin, e.g. native KLH, to obtain subunits, nanofiltrating thesubunits so obtained using filters removing viruses, preferablynanofilters with a pore size between 15 and 35 nm, more preferablybetween 20 and 25 nm and reassociating the subunits obtained afternanofiltering to obtain the synthetic hemocyanin, preferably thesynthetic KLH.

In a preferred embodiment, the subunits are immunocyanin. Morepreferably, the subunits are smaller than 800,000, 500,000, 400,000,350,000 or 300,000 Dalton, respectively. Most preferably, the subunitsare between 300,000 and 500,000 Dalton.

In this aspect of the invention, the dissociation of hemocyanin intosubunits is effected by applying a pH of between 8 and 10, preferably 9and 10. Preferably, the dissociation takes place at an alkaline pHbetween 8 and 10, preferably 9 and 10 under removal of the bivalentcations calcium (Ca⁺⁺) and the magnesium (Mg⁺⁺). Removal of Ca⁺⁺ andMg⁺⁺ may be effected by adding a chelat forming agent, e.g. EDTA. Underthese conditions, native KLH dissociates into subunits. It has beenfound that the dissociation is reversible, i.e. the subunits can bereassociated by re-establishing a neutral pH value (between 6 and 9,preferably between 7 and 8) to a heterogeneous mixture of didecamers andmultidecamers, preferably if Ca⁺⁺ and Mg⁺⁺ ions are added. A mostpreferred embodiment for dissociating hemocyanin such as native KLH, isas follows: The hemocyanin is stabilized in a stabilization buffer at aneutral pH including Ca⁺⁺ and Mg⁺⁺ ions, preferably a buffer includingTRIS/HCL at a pH between 7 and 8. In this buffer, the hemocyanin isstill in its native form without denaturating. An alkaline buffer isadded which is in the range between 8 and 10, preferably 9 and 10. Mostpreferably, the temperature is below 10° C., most preferably below 5°C., especially between 2 and 8° C. Preferred alkaline buffers compriseglycine and NaOH. Other preferred buffers comprise TRIS/HCl buffer pH8.9, TRIS/HCl buffer pH 8.9 plus EDTA, Sodium phosphate buffer pH 8.0,Ammonium carbonate buffer pH 8.0, Sodium bicarbonate buffer pH 10.1,Sodium bicarbonate buffer pH 9.5, NaCl and/or EDTA may be added to thebuffers. Typical buffer concentrations are 1-100 mM, preferably 2-50 mM,more preferably 10-20 mM. If EDTA is added, it is used in aconcentration of 1/10 to 1/2 compared to the buffer concentration. Sameconcentrations as for EDTA are contemplated for NaCl, if added. Thebuffer may include EDTA and/or NaCl.

The so-obtained solution of subunits (or immunocyanin solution) is keptat the alkaline pH (between 8 and 10, preferably 9 and 10) and can bestored for more than one month, more than two months, most preferablymore than three months, at a temperature below 10° C., more preferablybetween 2 and 8° C.

The subunits, e.g. immunocyanin, may be freed from a viralcontamination. Nanofiltration was previously shown to effectively removevarious viruses from protein solutions. However, the present inventorsfound that due to the enormous size and aggregation behaviour of KLHisomers, nanofiltration cannot be applied for KLH in its native form.The shift of the molecular weight of native KLH from more than 8 millionDaltons to a molecular weight of less than 500,000 Daltons, typically toa uniform molecular weight of approximately 400,000 Daltons of the KLHsubunits (immunocyanin), however, created the basis for the virusremoval realized by the present inventors. Nanofiltrations can be madewith commercially available nanofilters. Typically, these filters have apore size of 15-35 nm, more preferably between 20-25 nm. Such filtersare commercially available as filter capsules, for example Planovafilter capsules. In one embodiment, such nanofilters are a single useunit. The nanofilters may utilize a low protein binding hollow-fibremicroporous membrane constructed of naturally hydrophilic cuprammoniumregenerated cellulose with a narrow pore distribution. A wide range ofeffective surface areas may be applied between 0.001 m² up to 4 m²,preferably between 0.01 m² and 0.3 m².

The present inventors found that applying nanofiltration to hemocyaninsubunits (immunocyanin) under typical conditions of commercialnanofilters was not very effective. Typically, proteins are nanofilteredby employing a protein suspension of solution and pumping the proteinsuspension of solution with a constant flow rate between 0.01 and 10ml/minute, more preferably between 0.1 and 1 ml/minute into the filterso that the nanofilter surface is perpendicular to the flow direction.Such an approach is effective in virus removal. Unfortunately, due tothe high molecular weight of KLH subunits (immunocyanin), the virusfilters retain not only virus, but also high amounts of protein. Theclassical virus filtration with a flow perpendicular to the filtersurface is also known as “dead-end filtration”. The present inventorsfound that dead-end filtration is not preferred for virus removal inimmunocyanin, or hemocyanin subunits. Hemocyanin, even afterdissociation, cannot be nanofiltrated without a severe loss of protein.Example 2 compares “dead-end filtration” with the preferred mode offiltration of the present invention described herein below. Dead-endfiltration leads to a loss of protein of more than 40%, more than 60% oreven more than 80%.

Accordingly, there was a need for the provision of an improved andmodified virus filtration approach. The present inventors found that thesame nanofilters as described above need to be handled in a new andmodified manner: The protein suspension or solution needs to be pumpedwith a flow parallel to the membrane surface. The flow rate may bebetween 0.01 and 100 ml/minute, preferable between 0.1 and 100ml/minute, more preferably between 1 and 70 ml/minute. The pressureapplied to the protein solution or suspension is lower than 0.1 MPa,preferably lower than 10 kPa. The protein solution or suspension ispumped or flow over the membrane surface, preferably in a repeatedmanner, more preferably, under the addition of more protein solution orsuspension containing hemocyanin subunits so that a cycle including araw material flow is established. The nanofiltration takes placepreferably in an alkaline buffer, most preferably in the alkaline bufferused for dissociation. The starting material flown over the filter ispreferably in a concentration range between 0.1 and 10 mg/ml, morepreferably between 0.1 and 1 mg/ml, most preferably between 0.3 and 7mg/ml. The protein yield after filtration is more than 80%, morepreferably more than 90%, more preferably more than 93%.

The present inventors have established that with this nanofiltrationapproach, herein denominated “Cross-flow” filtration with flow directionparallel to the membrane surface of the nanofilter, almost quantitativeprotein purification is possible. Accordingly, the filtration issuitable for production of commercially relevant hemocyanin subunits orimmunocyanin.

The present inventors also established that the virus load can besufficiently reduced. With the methods of the present invention,preferably with cross-flow filtration, at least 99.9% of viruses areremoved from the protein material. The so-called log reduction factor isa measure for the virus removal. The log reduction factor (LRF) is theamount of virus removed from the initial protein solution formulation,i.e. protein or protein suspension, expressed on a logarithmic scale(dec log scale). An LRF of 1 means that 90% of viruses are removed, 10%are retained. An LRF of 2 means 99% of viruses are removed, 1% isretained. An LRF of 3 means 99.9% of viruses are removed, 0.01% areretained. With the cross-flow filtration of the present invention, atleast an LRF of 2 or more is obtained, more preferably an LRF of 3 ormore is obtained. More preferably, an LRF of 4 or more is obtained.

The proof of concept is shown in Example 3 (feasibility study forcross-flow filtration). In this experiment, a 20 nm Planova filter witha filter surface of 0.12 m² was employed. The flow rate was 50ml/minute. A total virus load of 10,620 (0.5% of the proteincomposition) was added. The virus employed for test purposes was PPV,one of the smallest viruses (diameter of 20 nm). A protein yield of morethan 97% was achieved (4,662.4 g of purified protein compared 4,814.9 gpre-filtrate). LRF was 3.14+/−0.32.

The obtained filtrate may be used for therapeutic purposes (immunocyaninpreparations). Accordingly, a method of producing immunocyanincomprising the steps of dissociating native hemocyanin to obtainsubunits and nanofiltering the subunits so-obtained through a filterwith a pore size between 15 and 35 nm is provided by the presentinvention. Preferably, the filtration is a cross-flow filtration. Morepreferably, the amount of an obtained protein is more than 60%, morethan 70%, preferably more than 80%, more preferably more than 90%, mostpreferably more than 93% of immunocyanin or hemocyanin subunits.

In another embodiment, the immunocyanin or hemocyanin subunits arereassociated after the nanofiltering to obtain a “synthetic” hemocyanin,preferably “synthetic” KLH. The reassociation is effected byre-establishing a neutral pH value. The protein suspension or proteinsolution is reassociated to a heterogeneous mixture of didecamers ormultidecamers by shifting the pH to the range between 6 and 9,preferably between 7 and 8. In a more preferred embodiment, thereassociation is effected by adding Ca⁺⁺ and Mg⁺⁺ ions. More preferably,the amount of Ca⁺⁺ and Mg⁺⁺ ions is lower than 0.5 M, each. Morepreferred, buffers such as 0.05-0.1 M TRIS/HCl buffer pH 7.4 areemployed, optionally together with between 0.05 M and 0.2 M MgCl₂,and/or between 0.05 M and 0.2 M CaCl₂, and/or between 0.15 M and 0.3 MNaCl. Other buffers are glycine/NaOH pH 7.4, or sodium phosphate, pH7.4.

Accordingly, the present invention in one embodiment provides a methodof producing synthetic KLH or synthetic hemocyanin comprising the stepsof dissociating native KLH to obtain subunits, nanofiltering thesubunits so-obtained using filters with a pore size between 15 and 35 nmand reassociating the subunits obtained after nanofiltering to obtainthe synthetic hemocyanin or synthetic KLH. Preferably the filtration isa cross-flow filtration, more preferably a cross-flow filtration asdescribed above. Typically, the amount of obtained protein is more than60% per amount of native KLH. More preferably, the obtained (syntheticKLH or synthetic hemocyanin) is more than 70%, more preferably more than80%, most preferably more than 90% or 93% per amount of native KLH.

The present invention in a third aspect provides the hemolymph obtainedby the method of the first and/or second aspect, the hemocyanin or thehemocyanin subunits obtained by the methods of the second aspect of theinvention. This aspect includes the provision of immunocyanin, which isa mixture of subunits of a hemocyanin in its naturally occurring ratio.

In a forth aspect, the hemolymph, the hemocyanin or the hemocyaninsubunits of the third aspect for use as a medicament are provided.

This aspect also covers a pharmaceutical composition comprising thehemolymph, the hemocyanin or the hemocyanin subunits of the third aspect

The pharmaceutical compositions or medicaments are e.g. for use in thetreatment of cancer, preferably bladder cancer, or as an immunostimulantor carrier.

According to a fifth aspect hemocyanin subunits are provided, which arethe result of a selective dissociation of hemocyanin produced accordingto the second aspect.

DETAILS OF THE INVENTION

In a first aspect of the invention a method for the preparation of lowendotoxin/low bioburden Hemolymph Sera from molluscs such as Megathuracrenulata in commercial quantities is provided.

In one embodiment, the present invention is directed to the preparationmethod of a pharmaceutical grade starting material derived frommolluscs, preferably keyhole limpets. The low endotoxin/low bioburdenquality of Hemolymph Sera is reached by applying a specific quarantineprocedure to limpets from natural or aquaculture source. The proprietarydesign of the Quarantine Aquaria System leads to significant reductionof biological contamination i.e. bacteria, endotoxins and viruses.

One key point of this first aspect of the invention is the treatment ofmolluscs in a quarantine aquarium system. The animals are kept underspecific temperature conditions and/or the quarantine aquaria includemeans for protein removal such as a centrifugal protein skimmer and/orone or more biofilters.

Preferably, artificial seawater is used in the aquarium and morepreferably, a rapid circulation of artificial seawater is employed totreat the molluscs. The current in the aquarium may imitate a surf zoneat the sea. Biological contaminants may removed by extensive foamingand/or bio-filtration of the quarantine water.

The Aquarium system of the first aspect leads to the reduction of thebiological contaminants. It removes excrements effectively and therebyleads to the removal of bacteria and bacterial endotoxins. During thetreatment in the aquarium, the animals are preferably not fed, whichagain minimized the content of organic ingredients and leads to areduction of contaminations.

Conductivity, pH-value, and the redox potential of the sea water arepreferably controlled and measured permanently.

The water temperature in the natural environment of the keyhole limpetsis between 10 and 20° C., preferably between 12-16° C.; thereforecooling of the water in the aquarium is required. For that purpose aheat exchanger may located in the reservoir. The water temperature ismonitored by temperature sensors in the basin, controlling cooling unitsfor the set temperature target (14° C.±2° C.).

The water in the aquarium is preferably artificial sea water, i.e. waterwhich is controlled in conductivity and pH, and preferably redoxpotential and more preferably in addition salt content to resemble seawater. For example, the conductivity value is between 46-52 ms/cm andthe pH between 7.5-8.5. In one embodiment, the density may range from1.020 to 1.030. A redox potential>100 is preferred.

Animals' release for puncturing requires one, preferably two, morepreferably three, more preferably four, more preferably five, morepreferably six, more preferably seven days, more preferably 10 days,most preferably 13 days of animal holding in the aquarium.

The in-house animal quarantine procedure of the invention aids inreducing the bio-burden. E.g. the animal coliform content can be reducedupon culturing under quarantine conditions.

Upon quarantine, the animals are punctured at their pedal blood sinusunder cold narcosis.

A second key point of the first aspect of the present invention,accordingly, is the procedure for puncturing the molluscs. Before thepuncture (“hemolymph extraction”), the molluscs are removed from theaquarium, may be examined visually, are preferably washed, andtransferred into a clean room facility.

The animals are preferably transferred into a clean room facility, wherethe animals are rinsed using hemolymph isotonic solution (HIS), aproprietary Sodium Chloride solution whose salt concentration isisotonic with animal sera.

The animals are weighed, and placed on pre set puncture racks.

During this operation preferably care is taken not to cause internalinjuries, especially to the intestinal system, for two reasons: first toavoid contamination of the product with fecal matter and secondly toavoid animal death. If accidental injury of the intestinal track arises,as indicated by fecally contaminated hemolymph sera, the material isdiscarded.

After disinfecting, a puncture may be made in the hind third of the baseof the foot with a needle that comprises a lumbal cannula. An inlet ofthe cannula may be stuck into the foot muscle and may be pushed infurther until the pedal sinus is reached. Neither the buccal sinus northe cardiac sinus is preferably punctured.

In one embodiment, upon completion of the blood extraction, sterileisotonic solution is injected, preferably through the cannula, andliquid leaking out is examined to determine if the blood sinus has beenreached, which is indicated by blue fluid.

In a preferred embodiment of the present invention, 10 to 60 ml, morepreferably 30-50 ml or 10-20%, preferably 12-15% of the body weight ofthe animal, of hemolymph is collected in a sterile centrifuge tube.

In one embodiment, the hemolymph that has been withdrawn is replaced byHIS solution.

The hemolymph is preferably refrigerated between 2 and 8° C. and may bepooled.

The animals are preferably transferred back, washed and returned to thein-house recovery aquaria tanks. The animals may be monitored for 1 to 4days and returned to their natural. This method permits the molluscs tobe returned to the ocean alive.

According to a third key point of the first aspect, the obtained bloodmay be purified and sterilized by 0.2 μm membrane filtration.

Before pooling, the hemolymph fractions must have been shown tocorrespond to the specifications of the IPC (in process control)performed on the single samples. The cold hemolymph fractions are pooledin a sterile disposable bottle and mixed well, while frothing isavoided.

In a second aspect of the invention a method for the preparation of lowendotoxin/low bio-burden hemocyanin or hemocyanin subunits from molluscssuch as Megathura crenulata in commercial quantities is provided.

Preferably, the method is capable of providing biological safe, virusfree molecular standardised hemocyanin, e.g. KLH, or hemocyaninsubunits, e.g. KLH subunits in commercial quantities.

The method comprises the isolation of hemocyanin from Hemolymph Sera,preferably via direct chromatography, more preferably via ion exchangechromatography.

The method preferably comprises a step of dissociating hemocyanin intothe hemocyanin subunits.

The method may also comprise a step of purifying hemocyanin subunits.

The method preferably also comprises a step of nanofiltration. In thisstep viruses may be separated from the hemocyanin subunits, i.e. thestep is performed in order to remove potential virologicalcontamination.

The method preferably also comprises a step of reassociating thehemocyanin from the subunits.

Hemolymph Sera based on its' origin from a marine mollusc contains apartfrom hemocyanin and other serum components high levels of SodiumChloride and other minerals. The conductivity of Hemolymph Sera based onits high salt content is on average around 50 ms/cm. To achievequantitative binding of hemocanin, e.g. of KLH, the conductivity has tobe reduced to <20 mS/cm.

In order to reduce the conductivity as described above the HemolymphSera may be partially desalinated by suitable methods such as gelfiltration, electrodialysis, diafiltration or dilution. The removal ofsalts leads to precipitation of the other serum components i.e proteinand carbohydrates. The precipitate may be removed by low speedcentrifugation, depth filtration or membrane filtration (0.8 u. 0.45μm).

Subsequently the colloidal dissolved high molecular weight KLH may beisolated by chromatography procedures i.e. ion exchange chromatography,which may then be followed by dissociation and purification of subunits.

Two preferred methods of dissociation of native hemocyanin, such as KLH,are possible: In situ dissociation on an ion exchange capture column ordissociation by Diafiltration.

The obtained hemocyanin subunits may be purified by an additional ionexchange chromatography step and finally polished by gel filtration.

The native, oxygen-binding hemocyanin protein in one embodiment ispurified from the hemolymph by Ion Exchange Chromatography. Thehemocyanin is be bound to an anion exchanger and then dissociated on thecolumn into the KLH subunits (immunocyanin) in alkaline (pH 7 to 10,preferably 8.6 to 9.6) buffer. The immunocyanin is recovered from thecolumn by means of salt gradient elution. The resulting immunocyaninsolution may be desalinated and concentrated bydiafiltration/ultrafiltration. The concentrated immunocyanin solutionmay be subsequently purified by a further ion exchange chromatographystep.

In another embodiment, the native, oxygen-binding hemocyanin protein ispurified from the hemolymph by Ion Exchange Chromatography. Thehemocyanin is bound to an anion exchanger and then recovered from thecolumn by means of salt gradient elution. The resulting hemocyaninsolution is desalinated, concentrated and dissociated into the KLHsubunits (immunocyanin) by means of diafiltration, dialysis orultrafiltration in alkaline (pH 7 to 10, preferably 8.6 to 9.6) buffer.Finally the immunocyanin solution may be concentrated followed by afurther ion exchange chromatography step.

In another embodiment, the native, oxygen-binding hemocyanin protein ispurified from the hemolymph by Ion Exchange Chromatography. Thehemocyanin is bound to an anion exchanger and then recovered from thecolumn by means of salt gradient elution. The hemocyanin of theresulting hemocyanin solution is isolated by means ofultracentrifugation. Subsequently the resulting hemocyanin pellets aredissolved and dissociated into the KLH subunits (immunocyanin) inalkaline (pH 7 to 10, preferably 8.6 to 9.6) buffer. Finally theimmunocyanin solution may be concentrated followed by a further ionexchange chromatography step.

In order to achieve dissociation of hemocyanin, in general, thehemocyanin may be dissolved in dissociation buffer (pH 8 to 10,preferably 8.6 to 9.6, preferably devoid of Ca⁺⁺ and Mg⁺⁺). This createsalkaline conditions, which lead to dissociation of the native hemocyaninmolecule into its subunits.

The immunocyanin solution may be concentrated. Before a finalpurification (polishing), e.g. by gel filtration, the immunocyaninsolution may be concentrated to a protein content of 20 mg/mL (±2.5mg/mL), e.g. by ultrafiltration. For this purpose, low protein bindingpolysulfone or polyether sulfone membranes (separation limit: 30,000Dalton; filter area: ≧700 cm²) mounted in a stainless steelultrafiltration unit are preferably used. After ultrafiltration, theconcentrated immunocyanin solution is filtered, e.g. through a 0.22 μmmembrane filter.

The concentrated immunocyanin solution may then purified, e.g. by middlepressure liquid chromatography through a gel filtration column.

A preferred column is Superose® 6 (preparative grade; composed of highlycross-linked porous agarose beads); bead size 20-40 μm, fractionationrange 5,000-5,000,000 Da. As eluent an elution buffer (pH 8-10, devoidof Ca⁺⁺ and Mg⁺⁺) may be used. The concentrated immunocyanin solutionmay be loaded onto the column under aseptic conditions. The mainimmunocyanin peak at molecular weight 400,000 is collected. Theimmunocyanin fraction is preferably immediately cooled to +2-8° C. andfiltered, e.g. through a 0.22 μm membrane filter.

Due to the origin of native hemocyanins such as KLH a virological riskby human pathogens exist. To guarantee biological safety the downstreamprocess of biologicals preferably contain steps for inactivation orremoval of potential virus contamination. The available inactivationmethods were tested on KLH and found to be not suitable for hemocyaninsbecause of their damaging effect on the KLH preparations.

According to the present invention nanofiltration is preferably used toobtain purified hemocyanin subunits, e.g. KLH subunits, i.e. for removalof potential virus contamination.

In this step a suitable virus filter membrane, which has no influence onthe content and biochemical, chemical and physical characteristics ofhemocyanin subunits, e.g. KLH subunits may be selected. Unfiltered andfiltered hemocyanin subunits are compared in a comparability study inorder to show that the subunits are functionally intact. VirusValidation Study may be performed to show the effect of virus filtrationof hemocyanin subunits on the removal of model viruses with differentsizes.

In order to demonstrate the safety of pharmaceutical proteins derivedfrom biological sources it is mandatory for the manufacturer of suchproducts to demonstrate the effective inactivation and/or removal ofpathogenic viruses during the manufacturing process. Usually, this isdone by the deliberate spiking of a down-scaled version of themanufacturing process with relevant and/or model viruses.

According to the present invention, the removal of at least MurineLeukemia Virus, Pseudorabies Virus, Reo Virus Type 3, and PorcineParvovirus by nanofiltration is performed. In order to test the removal,a test sample will be spiked with the viruses at defined titers and thensubjected to nanofiltration. Samples may be withdrawn from the spiked,prefiltered test sample as well as from the nanofiltrate and monitoredfor virus by endpoint titration and by bulk analysis, respectively.

Removal of viruses is preferably performed in order to reduce the virustiter by 50%, preferably 60%, more preferably 70%, more preferably 80,more preferably 90, more preferably 99%, most preferably 99.9%.

Relevant viruses representing potential contaminants of hemocyaninproducts are as follows:

Indicator Model Virus Taxonomy Genome Structure Size/nm Stability cellline Hepatitis A virus Picornaviridae ssRNA non-enveloped 25-30 mediumto high FRhK-4 (HAV) Bovine Viral Diarrhoea Flavivirus ssRNA enveloped40-60 low MDBK Virus (BVDV) Porcine Parvovirus Parvoviridae ssDNAnon-enveloped 18-24 very high PK13 (PPV) Simian Virus 40 PapovaviridaedsDNA non-enveloped 40-50 high Vero (SV40)

Hepatitis A Virus (HAV)

non-enveloped, small (25-30 nm), single-stranded RNA virus (ATCCVR-1402) with a medium to high resistance to physico-chemicalinactivation. Hepatitis A belongs to the Picornaviridae family, whichalso includes EMCV and Poliovirus. This virus is a potential contaminantof human blood and plasma and therefore should be used where possible instudies. However, the presence of neutralizing antibodies to this virusin blood and plasma products means that its use is limited to situationswhere this problem does not occur.

Porcine Parvovirus (PPV)

unenveloped, small (˜18-25 nm), single-stranded DNA virus (provided byOctapharma AG, Frankfurt, Germany) with a high resistance tophysico-chemical inactivation. It therefore provides a severe test forthe clearance and reduction capacity of the downstream process system.The human Parvovirus B19 virus can be present at high titres in humanplasma, and therefore PPV can be used as a model for B19 in thevalidation of human plasma derived products. There are also reportedincidences of contamination of recombinant products with Parvovirusessuch as Murine Minute virus, and PPV can be used as model for this classof virus.

Bovine Viral Diarrhoea Virus (BVDV)

enveloped, medium-sized (˜40-60 nm), single-stranded RNA virus (ATCCVR-534) with a medium resistance to physico-chemical inactivation. BVDVbelongs to the Flaviviridae family which also contains Hepatitis C andHepatitis G viruses. BVDV is therefore a suitable model virus whereHepatitis C or G is of concern, particularly in a product derived fromhuman blood, and also for other Flavivirus and Togavirus contaminants,for example where bovine derived material is used.

Simian Virus40 (SV40)

unenveloped, small (˜40 nm), double-stranded DNA virus (ATCC VR-305)with high resistance to physico-chemical inactivation. SV40 provides asevere test of the downstream process and its capacity toremove/inactivate viruses. This virus acts as a model for otherresistant unenveloped viruses which may be present as contaminants inthe starting material, and is a model for papilloma and polyoma viruscontaminants.

In the present invention, at least HAV, BVDV, and SV40 are removed,preferably with LRF of more than 2, 3, 4, 5 or 6 for each virus. PPV maybe removed with LRF of more than 2, 3 or 4. Alternatively, or inaddition, viruses as follows are also removed from the hemocyanin orimmunocyanin of the present invention: Murine Leukemia Virus (MuLV),Pseudorabies Virus (PRV) and Reovirus type III (Reo III). These virusesare also removed with LRFs of at least 2 or 3 for each virus.

The stabilisation of virus filtered hemocyanin subunits may be performedby means of lyophilisation. Protein solutions (especially high molecularweight proteins) are in general not stable in the long term. Duringstorage protein precipitation together with loss of activity occurs.Storage in stabilising buffer systems or in applicable solutions forpharmaceutical use under refrigerated conditions did not lead to KLHpreparations satisfying stability required for pharmaceuticals (2-3years). Lyophilisation of high molecular weight proteins under retentionof their full biological activity is only possible with a suitablemixture of excipients. According to the present invention, proteinstabilizers, e.g. lactose, mannitol, sucrose, etc may be used. Thesestabilizers may be added to the purified immunocyanine solution assolutions with concentrations of 100-700 mg/ml and with a volume of 0.1ml to 1.0 ml per 1 mg of protein each.

The lyophilised KLH subunits are proofed for their full biologicalactivity and their molecular intactness.

The Stabilisation of virus filtered hemocyanin subunits may also beperformed by means of desalination. Desalination by diafiltration withwater for injection leads to a salt free highly concentrated (20 mg/ml)hemocyanin subunit solution in water with unexpected long term stabilityunder refrigerated conditions (1-2 years). The salt free hemocyaninsubunit solution is the ideal carrier for the manufacturing ofconjugated vaccines.

Means and methods for desalination are known by the persons skilled inthe art. Typically desalination is performed as long as the conductivityof the filtrate in an iterative filtration process is ≧10 μS/cm or theconductivity of the retentate is ≧150 μS/cm.

The salt free hemocyanin subunits are proofed for their full biologicalactivity and their molecular intactness.

Hemocyanin subunits may be used as a mixture in the ration present innative hemocyanin (“immunocyanin”) or single subunits may be separatedand used upon isolation.

In order to obtain “synthetic” hemocyanin, the reassociation of thesubunits has to be performed, i.e. the refolding of virus filtered KLHsubunits.

The size of hemocanin oligomers, e.g. of KLH didecamers (approx. 35 nm),is situated in the same size of large viruses. The described isolationand purification methods (ion exchange chromatography and gelfiltration) for native KLH do not lead to the required reduction factorsof potential virological contamination according to establishedguidelines on virological safety of biological products derived fromanimal sources. Dissociation into the hemocanin subunits reduces thesize, in the case of KLH to approx. 400,000 Dalton, and makes theprotein accessible to nanofiltration. Refolding is performed with bufferexchange, e.g. by diafiltration or dialysis under reassociationconditions (pH 7-8, Ca⁺⁺, Mg⁺⁺). The reassociated hemocanin may furtherbe purified by gel filtration.

In order to achieve reassociation a reassociation buffer is added to themixture of hemocyanin subunits.

High molecular weight KLH is manufactured from concentrated immunocyaninsolution by buffer exchange to reassociation conditions, pH 7-8, Ca⁺⁺,Mg⁺⁺ and preferably by a concentration step. Both may be achieved by oneor more up to a series of ultrafiltration steps using a polysulfonemembrane with a nominal separation limit of 50,000 Da.

Further embodiments of the invention are:

1. A method for the preparation of hemolymph sera from a mollusc:

comprisinga) puncturing the pedal blood sinus of the mollusc under cold narcosis.

2. The method of embodiment 1, wherein the mollusc is Megathuracrenulata.

3. A method of any of embodiment 1 or 2, wherein before puncturing themollusc is kept under specific quarantine conditions, wherein no organicfeed is supplied and/or the water in the aquarium system is purified byremoving biological contaminants.

4. The method of any of embodiments 1 to 3, wherein the blood obtainedupon puncturing is sterilized by 0.2 μm membrane filtration.

5. The method of isolating native hemocyanin or a mixture of subunitsthereof comprising

a) providing hemolymph sera obtained upon performing of any ofembodiments 1 to 4,b) isolating the hemocyanin from hemolymph sera by employing directchromatography,

6. The method of embodiment 5, wherein the direct chromatography is anion exchange chromatography.

7. The method of any of embodiments 5 or 6 further including a step ofdissociating the hemocyanin into subunits of the hemocyanin oligomer,purifying the KLH subunits and optionally re-associating the subunitsinto the oligomeric form of the hemocyanin.

8. The method of embodiment 7, wherein the purification is performedemploying nanofiltration in order to remove potential biologicalcontamination.

9. The method of any of embodiments 7 or 8, wherein the re-associationtakes place in a diafiltration step or a gel filtration step.

10. The method of any of embodiments 5 to 9 wherein the hemocyanin isadmixed with a stabilizing puffer system for long-term storage.

11. The hemolymph obtained by any of embodiments 1 to 4, the hemocyaninor the mixture of hemocyanin subunits obtained by any of embodiments 5to 10.

12. The hemolymph obtained by any of embodiments 1 to 4, the hemocyaninor the mixture of hemocyanin subunits obtained by any of embodiments 5to 10 as a medicament.

14. A pharmaceutical composition comprising the hemolymph obtained byany of embodiments 1 to 4, the hemocyanin or the mixture of hemocyaninsubunits obtained by any of embodiments 5 to 10.

15. The hemolymph of embodiment 11 or the hemocyanin of embodiment 12for use in the treatment of cancer, preferably bladder cancer, or as animmunostimulant or carrier.

16. A method of providing one or more hemocyanin subunits comprisingperforming the method of any of embodiments 1 to 4 and the method of anyof embodiments 5 to 10 and a step of selectively dissociating the soobtained reassociated hemocyanin.

17. The one or more hemocyanin subunits obtained by the method ofembodiment 16.

18. The pharmaceutical comprising the one or more hemocyanin subunits ofembodiment

17. The one or more hemocyanin subunits of embodiment 17 may be for usein the treatment of cancer or as an immunostimulant.

19. The use of cross-flow filtration for removing viruses from proteinformulations.

20. The use of embodiment 19, wherein the protein formulation includes aprotein of between 100,000 and 1,000,000 Dalton.

21. The use according to embodiments 19 and 20, wherein the cross-flowfiltration employs filters of a pore size between 15 and 35 nm.

EXAMPLES Example 1: Quarantine of Megathura Crenulata

Since the start of the production activities at our facility inCarlsbad, Calif. in February 2002, we have collected 13 batches ofanimals at various time periods. The collection site ID is the zonenumber as defined by the Southern California Fisheries Chart (SCFC).

The weather condition in California during the collection of animals forbatches MC-001 to MC-013 were not unusual. However, during the morerecent batch of animals collected, MC-014, California had experiencedunusual rainy conditions prior to animal collection, however, on the dayof animal receipt we experienced no rains.

It may be pointed out that the incorporation of test methods has evolvedsince we started the activities at our facility. The fecal coliformtesting on animals, toxic substance, DDT and PCB testing was initiatedwith lot# MC-002. The pH and Conductivity testing has been done for alllots, the nitrate, nitrite and ammonia testing was initiated on the seawater sample with lot# MC-006.

The pH and conductivity of sea water has a range of 8.0 to 8.3 and 45.0to 52.4 respectively. The nitrate, nitrite and ammonia content rangedfrom 0 to 80 ppm, 0 to 0.25 ppm and 0.25 to 1.0 ppm respectively, thehigh end of the range corresponds to the values for lot MC-014, which,as has been noted was collected post heavy rains. The fecal coliform ofsea water samples were <2 MPN/100 mL for samples collected from MC-002to MC-013 and were 29, 11 and 49 MPN for the most recent lot MC-014corresponding to the three water samples “0”, “50” and “100”respectively.

The DDT and PCB test results indicate that they are below the detectionlimit of the respective assays. It appears that this may not be an issuein the collection site 718 and 719 where the animals were collectedfrom.

The animal fecal coliform data suggests that the fecal coliform weregenerally <18 and a maximum of 20 MPN/100 grams for the lot# MC-002 toMC-013, however for MC-014 the values were very high, 3,500 MPN/100gram. These results in conjunction with the fecal coliform in thesurrounding sea water suggest that these animals tend to concentrate thefecal coliform.

On receipt of the laboratory results for fecal coliform in animals, wedecided to send samples of animals from our quarantine tanks, 3 animalswere taken from Tank Q1 and 3 from Tank Q2, on Jan. 19 2005. The animalswere received into our tanks on Jan. 6 2005 and therefore were presentin our tank water for 13 days prior to testing. We initiated thistesting to determine if the quarantine procedures that we haveincorporated into our manufacturing schedule would have an effect onreducing the animal fecal coliform content. The copy of the test reportis attached to this report as an attachment. The results indicate thatthe animal fecal coliform is <18 MPN/100 gram. These results are veryencouraging and suggest that the procedures in place are effective inreducing the animal coliform content, if any are present as with thisLot MC-014.

The analysis of the data relating to animals and sea water suggest that:

The pH and conductivity of sea water provide information on the naturalsea water conditions and would be useful to compare with our artificialsea water prepared in-house. We currently have a specification forartificial sea water as 7.5 to 8.5 and conductivity 46-52 ms/cm. Thesespecifications seem to match well with the ranges for natural seawater.

The toxicological screening for sea water from collection site wasinitiated based on advice from Dr. Robert Mooney, Merkel & Associates,used as an external animal health inspector for the first three lots ofanimal received, namely MC-001 to MC-004. The results to date suggestthat DDT and PCB's are not an issue for the area where the limpets arecollected from and released into.

The in-house animal quarantine procedure seems to aid in reducing theanimal coliform content and is a very useful procedure. Currently,animals release for manufacture require a minimum of seven days fromstart of quarantine, the animal fecal coliform data for MC-014 wasobtained on animals after 13 days of animal holding in our tanks. It maybe necessary to initiate a more systematic investigation into the lengthof quarantine and reduction of fecal coliform and determine if thecurrent set specification of seven days is sufficient. Such studies tobe initiated after collection of animals from waters with high coliformas was the case with MC-014.

Example 2: Direct Chromatographic Isolation of Native Hemocyanin fromHemolymph Sera

Hemolymph Sera based on its' origin from a marine mollusc contains,apart from hemocyanin and other serum components, high levels of SodiumChloride and other minerals. The conductivity on average is around 50ms/cm. Under those present conditions KLH cannot be bound to IonExchange resins. To achieve quantitative binding of KLH the conductivityhas to be reduced to <20 mS/cm.

In order to reduce the conductivity as described above the HemolymphSera is partially desalinated by suitable methods such as gelfiltration, electrodialysis, diafiltration or dilution. The removal ofsalts leads to precipitation of the other serum components i.e proteinand carbohydrates. The precipitation is removed by low speedcentrifugation, depth filtration or membrane filtration (0.8 u. 0.45μm).

Subsequently the colloidal dissolved high molecular weight KLH isisolated by chromatography procedures i.e. IEX chromatography followedby dissociation and purification of subunits.

Dissociation of Hemocyanin Method 1:

The native, oxygen-binding hemocyanin protein is purified from thehemolymph by Ion Exchange Chromatography. The hemocyanin is bound to ananion exchanger and then dissociated on the column into the KLH subunits(immunocyanin) in alkaline (pH 9.6) buffer. The immunocyanin isrecovered from the column by means of salt gradient elution. Theresulting immunocyanin solution is desalinated and concentrated bydiafiltration/ultrafiltration. The concentrated immunocyanin solutionmay be subsequently purified by a further IEX chromatography step.

Method 2:

The native, oxygen-binding hemocyanin protein is purified from thehemolymph by Ion Exchange Chromatography. The hemocyanin is bound to ananion exchanger and then recovered from the column by means of saltgradient elution. The resulting hemocyanin solution is desalinated,concentrated and dissociated into the KLH subunits (immunocyanin) bymeans of diafiltration, dialysis or ultrafiltration in alkaline (pH 9.6)buffer. Finally the immunocyanin solution is concentrated followed bypurification with a further IEX chromatography step.

The hemocyanin obtained from Method 1/Method 2 is dissolved indissociation buffer (pH 9.6, devoid of Ca⁺⁺ and Mg⁺⁺). This createsalkaline conditions, which lead to dissociation of the native hemocyaninmolecule into its subunits. The entity of these subunits are calledimmunocyanin.

Method 3:

The native, oxygen-binding hemocyanin protein is purified from thehemolymph by Ion Exchange Chromatography. The hemocyanin is bound to ananion exchanger and then recovered from the column by means of saltgradient elution. The hemocyanin of the resulting hemocyanin solution isisolated by means of ultracentrifugation. The obtained pellets aredissolved in dissociation buffer. Finally, the immunocyanin solution isconcentrated and may be purified with a further IEX chromatography step.

Concentration of Immunocyanin Solution

Before final purification (polishing) by gel filtration, theimmunocyanin solution is concentrated to a protein content of 20 mg/mL(±2.5 mg/mL) by ultrafiltration. For this purpose, low protein bindingpolysulfone or polyether sulfone membranes (separation limit: 30,000Dalton; filter area: ≧700 cm²) mounted in a stainless steelultrafiltration unit are used.

After ultrafiltration, the concentrated immunocyanin solution isfiltered through a 0.22 μm membrane filter.

Purification (Polishing)

The concentrated immunocyanin solution is finally purified by middlepressure liquid chromatography through a gel filtration column.

Example 3: Nanofiltration of Purified KLH Subunits (Immunocyanin)

Due to the origin of native KLH exists a virological risk by humanpathogens. To guarantee biological safety the downstream process ofbiologicals should contain steps for inactivation or removal ofpotential virus contamination. Commercially available inactivationmethods were tested on KLH and found to be not suitable because of theirdamaging effect on the KLH preparations (pH reduction, heat treatment).

Nanofiltration was previously shown to effectively remove variousviruses from protein compositions. However, nanofiltration turned out tonot be useful for hemocyanins or native KLH, due to the molecular weightof native KLH from >8 Mill. Dalton. The filters could not discriminateviruses from protein, i.e. the protein is too big to pass the membraneof typical virus filters (pore size between 15 and 35 nm). A reductionto the uniform molecular weight of approx. 400,000 Dalton of KLHsubunits was affected. Several virus filters with different pore sizeshave been tested in a down scaled process.

Reference Example: Dead End—Filtration Protocol, Virus Filtration of KLHSubunits

A protein of approx. 400 KD in a concentration of approx. 5 mg/mlobtained from California see snail's blood has been filtered throughPlanova 20N, 0.001 m² in Dead-End modus with constant pressure of 2.0bar. The flow rate was 0.4 ml/min. The protein formulation was at a pHof 9.6 in a glycine/NaOH buffer. 1 g of starting material was applied.With dead-end filtration, 0.1 g of protein in a concentration with 0.5mg/ml was obtained, i.e. a protein yield of 10%. Also, a reduction ofthe starting amounts of the protein or the concentration of the proteinby a factor of 10 or more did not lead to different results. Also, thereduction of pressure or the increase of size did not lead to changes ofprotein yield.

Working Example: Cross-Flow Filtration Protocol; Virus Filtration of KLHSubunits

A protein of approx. 400 KD in a concentration of approx. 0.45 mg/mlobtained from California sea snails' blood has been filtered throughPlanova 20N, 0.12 m² in cross-flow modus with a constant pressure of0.16 bar. Formulation was in a buffer of glycine and NaOH at a pH of9.6. The amount of starting material was 5,000 g in a concentration of0.45 mg/ml. The protein amount obtained after nanofiltration was 4,688 gin a concentration of 0.42 mg/ml. This makes up to a yield of 93%.

This example demonstrates that contrary to dead-end filtration,cross-flow filtration enables the filtration of quantitative amounts ofhemocyanine protein upon dissociation into its subunits. Nanofilters ofa pore size between 15 and 35 nm can be employed which are sufficient toremove the smallest viruses known.

Example 4: Proof of Concept: Feasibility Study

This example demonstrates the suitability of cross-flow filtration forremoving small viruses of a diameter of the smallest viruses known. Inthis example, PPV was tested. PPV has a diameter of 20 nm and the viruswas spiked at a concentration of 0.5% per protein. Immunocyanin, aprotein of uniform molecular weight of approximately 400,000 Dalton ofKLH subunits was spiked with 0.5% PPV. The total virus load inprefiltered was 10,620. The protein amount after virus spiking was4,814.9 g. Nanofiltration was performed with Planova 20N nanofilters,0.12 m² in cross-flow mode. The flow-rate chosen was 50 mm/min with aconstant pressure of 0.28 bar. 4,662.4 g protein were retained in thefiltrate. The LRF for PPV was 3.14+/−0.32. Accordingly, the proteinamount was more than 93% with a virus removal of more than 99.9%.

This example shows that cross-flow filtration is suitable for thepreparation of KLH or KLH subunits in a virus free form on acommercially relevant scale with a yield of more than 90%.

Example 5: Stabilisation of Purified KLH Subunits by Means ofDesalination

Das Folgende würde ich noch kürzer zusammenfassen, so z.B.!

Principle

KLH BULK LIQUID salt-free is manufactured from purified immunocyanin bydesalination and concentration. Both are achieved by a series ofultrafiltration steps using a polysulfone membrane with a nominalseparation limit of 30,000 Da.

Preparation of the Desalination Batch

In order to minimize batch-to-batch variations during the desalinationprocess, purified immunocyanin solution is concentrated to animmunocyanin content of between 10 mg/ml and 40 mg/ml (±2 mg/ml) byultrafiltration. For this purpose, low protein binding polysulfone orpolyether sulfone membranes (separation limit: 30,000 Da) mounted in astainless steel ultrafiltration unit are used. Before use, the membranesare conditioned by recirculation with alkaline dissociation buffer at atemperature of +2-8° C. Flow is achieved by a peristaltic pump. Finally,the conditioning is tested by in-process control pH and bacterialendotoxins. Before starting the concentration process, theultrafiltration unit is checked for integrity.

Concentration

The immunocyanin solution is now transferred to the ultrafiltration unitand recirculated at +2-8° C. The concentration is controlled be weighingthe obtained ultrafiltrate. The maximal entrance pressure of theultrafiltration unit should not exceed 1 bar, preferably not 0.5 bar.The immunocyanin solution is recirculated until the calculated amount ofultrafiltrate has been collected. Finally, the concentrate is tested byin-process control pH value, osmolality, conductivity, immunocyanincontent.

Desalination

The concentrated immunocyanin solution (=concentrated desalinationbatch) is either desalinated by dilution 1+1 with water for injectionsat each ultrafiltration cycle or alternatively by adding the water forinjections employing constant volume wash procedure. The desalinationprocess is controlled by weighing the ultrafiltrate, the concentrateddesalination batch and testing of conductivity of the ultrafiltrate andthe desalination batch.

If the conductivity of the ultrafiltrate has reached <10 μS/cm or if theconductivity of the concentrated desalination batch is <150 μS/cm, thedesalination process is terminated and the immunocyanin content of thedesalination batch is determined in order to prepare the final batch ofKLH BULK LIQUID salt-free.

Preparation of the Final Batch of KLH BULK LIQUID Salt-Free

The final batch of KLH BULK LIQUID salt-free is prepared from theimmunocyanin concentrate by dilution to an immunocyanin content of 20mg/ml. For this purpose, the filtered immunocyanin concentrate isweighed. The required amount of water for injections is weighedaccurately and slowly added to the filtered immunocyanin concentrate.The solution is gently mixed, and a sample for in-process control isremoved pH value, density, osmolality, conductivity, immunocyanincontent.

The released solution is finally sterilized by filtration through a 0.22μm membrane filter directly into infusion bags.

They are stored at +2-8° C.

During filtration, samples for quality control are removed.

Example 6: Refolding of Virus Filtered KLH Subunits and FinalPurification Principle

High molecular weight KLH is manufactured from concentrated immunocyaninsolution by diafiltration (buffer exchange to reassociation conditions,pH 7-8, Ca⁺⁺, Mg⁺⁺) and concentration. Both are, e.g. achieved by aseries of ultrafiltration steps using a polysulfone membrane with anominal separation limit of 50,000 Da.

Concentration of the Purified Immunocyanin Solution

In order to optimize reassociation conditions, purified immunocyaninsolution is concentrated to an immunocyanin content of 20 mg/mL (±2mg/mL) by ultrafiltration. For this purpose, low protein bindingpolysulfone or polyether sulfone membranes (separation limit: 30,000 Da)mounted in a stainless steel ultrafiltration unit are used. Before use,the membranes are conditioned with elution buffer as follows. Theultrafiltration system is first rinsed with elution buffer at atemperature of +2-8° C., while the filtrate outlets are closed. Flow isachieved by a peristaltic pump. Finally, the elution buffer iscompletely removed from the system. A sample is removed from theretentate side for in-process control pH, bacterial endotoxins. Beforestarting the concentration process, the ultrafiltration unit is checkedfor integrity.

The immunocyanin solution is now transferred to the retentate bag of theultrafiltration unit. The recirculation is started, and theultrafiltrate is collected in a weighed beaker. The temperature is keptat +2-8° C. As during conditioning, the maximal entrance pressure of theultrafiltration unit should not exceed 1 bar. The immunocyanin solutionis recirculated until the calculated amount of ultrafiltrate has beencollected. The retentate is mixed, while the filtrate outlets areclosed, and a sample is removed for in-process control pH value,osmolality, conductivity, immunocyanin content.

Reassociation

In order to refold the KLH subunits a second ultrafiltration system withlow protein binding polysulfone or polyether sulfone membranes(separation limit: 50,000 Da) mounted in a stainless steelultrafiltration unit are used. Before use, the membranes are conditionedwith reassociation buffer as follows. The ultrafiltration system isfirst rinsed with reassociation buffer at a temperature of +2-8° C.,while the filtrate outlets are closed. Flow is achieved by a peristalticpump. Finally, the reassociation buffer is completely removed from thesystem. A sample is removed from the retentate side for in-processcontrol pH, bacterial endotoxins. Before starting the reassociationprocess, the ultrafiltration unit is checked for integrity.

For reassociation the ultrafiltration system is recirculated withreassociation buffer (between 2- to 10-fold volume of concentratedimmunocyanin solution) while the filtrate outlets are closed. Theconcentrated immunocyanin solution is slowly injected in therecirculated reassociation buffer. The temperature during the wholereassociation process is kept at a temperature of +2-8° C. Aftercomplete injection of the concentrated immunocyanin solution thereassociation batch is diafiltrated against between 2- and 10-fold ofreassociation buffer applying the constant volume wash procedure.Finally the batch is concentrated to a KLH content of 20 mg/ml. Afterreassociation, the concentrated KLH solution, is filtered through a 0.22μm membrane filter.

Purification of Refolded KLH by Gel Filtration

The concentrated KLH solution is finally purified by middle pressureliquid chromatography through a gel filtration column.

Biological Activity and Potency—Comparability with Native KLH

Native KLH and synthetic KLH obtained after reassociating according tothe method of the present invention were compared. Synthetic KLH andnative KLH were compared via CD-spectroscopy. Bands in CD-spectroscopywere identical.

The protein bands in SDS PAGE were identical when comparing syntheticand native KLH. In synthetic KLH, no protein fragments are found.

2-dimensional immunoelectrophoresis was also performed to comparesynthetic and native KLH. Anti-KLH1 and anti-KLH sera were used. Theimmunoelectrophoretic patterns were identical for both, native andsynthetic, KLH. Two precipitation maxima (one for KLH1 and one for KLH2)occur for both native and synthetic KLH.

Electromicroscopic investigations both, native and synthetic, KLH showthe typical decamers, didecamers, and tridecamers.

Native PAGE and densiometric tests show that both synthetic and nativeKLH include the typical protein bands. A ratio between KLH1 and KLH2between 0.9 and 1.0 for both, synthetic and native, KLH was obtained.

Example 9: Anion—Exchange Chromatography

For the isolation, purification and in situ dissociation of hemocyaninanion exchange chromatography was selected because of its isoelectricpoint (pI) at ˜pH 6 and the negative charge at pH>7.

Hydrophobic interaction separation was involved because of applyingdifferent conditions of those used in ion exchange chromatography (IEX).In this separation, a buffer with a high ionic strength, usuallyammonium sulfate, is initially applied to the column. The salt in thebuffer reduces the solvation of sample solutes thus as solvationdecreases, hydrophobic regions that become exposed are adsorbed by themedium.

In a first series of binding studies using hemocyanin solutions with lowsalt concentration different strong and weak anion exchange media packedin columns (bed volumes approx. 20-50 ml) as well as different HICcolumns were proofed on their binding capacity.

Strong and weak anion exchange media were selected due to their specificbinding properties and selectivity:

Poros® 50 micron media for perfusion chromatography (HQ—strong anionexchanger, functional group: quaternized polyethyleneimine; PI—weakanion exchanger, functional group: quaternized polyethyleneimine; D—weakanion exchanger, functional group: dimethyl amino alkyl groups DEAE).

The Poros® 50 micron media were used because of their robust chemicalstability, particle size of 50 μm, and the large pore structure (appr.6000 Å) which enables high flow rates without compromising capacity orresolution.

Q-Sepharose FF and HP (strong anion exchanger) with particle sizes of 90μm and 34 μm were also tested alternatively. The functional groupsexists of quarternary ammonium. Hydrophobic interaction chromatographytests were performed using a HiTrap HIC Selection Kit, which containsprepacked columns ready to use (HiTrap HIC Phenyl FF high sub 1 ml,HiTrap HIC Phenyl FF low sub 1 ml, HiTrap HIC Butyl FF 1 ml and HiTrapHIC Octyl FF 1 ml).

In summary no or only minor binding of hemocyanin could be obtained withthe hydrophobic interaction chromatography media. The Q-Sepharose FF andHP and the weak anion exchanger of the Poros® media (PI and D) showedonly less to medium binding. The highest binding capacity of approx. 20mg KLH per ml gel has been proofed with Poros® 50 micron HQ media.

Based on the obtained results the developmental work was continued inorder to purify KLH directly from the starting material Hemolymph Sera.

Hemolymph Sera based on its origin from a marine mollusc contains apartfrom hemocyanin and other serum components high levels of sodiumchloride and other minerals. The conductivity on average is around 50mS/cm. Under those present conditions KLH could not be bound to theselected Poros® 50 HQ media.

In order to achieve quantitative binding of KLH the hemolymph sera wasdiluted with TRIS/HCl buffer (pH 7.4, including CaCl₂, MgCl₂ and NaCl),so called IEXdilution buffer. The precipitation occurred during dilutionwas removed by prefiltration steps.

4 (four) dilution series were tested to determine the binding capacityof Poros® 50 HQ media for KLH (hemolymph sera diluted to approx. 25mS/cm, 20 mS/cm, 15 mS/cm and 10 mS/cm). Tests were performed on Poros®50 HQ columns (approx. 50 ml gel). The elution of KLH started at approx.27 mS/cm.

Flow rate sample: 5 ml/min

Flow rate elution: 10 ml/min

Elution gradient: 0.15 to 0.65 M sodium chloride, TRIS/HCl buffer (pH7.4, including CaCl2, MgCl2 and NaCl).

The binding studies showed that the hemolymph sera has to be diluted to<20 mS/cm to achieve quantitative binding of KLH.

Subsequently additional test runs with hemolymph sera diluted to approx.19 mS/cm were performed to optimize KLH binding addicted to the samplefeeding velocity. 4 (four) different sample flows (4.0 ml/min, 4.6ml/min, 5.2 ml/min and 8.0 ml/min) were tested under the conditionsdescribed above. The obtained results demonstrated the correlationbetween feeding velocity and binding capacity of the anion exchangermedia. With the lowest flow rate of 4.0 ml/min (corresponding to alinear flow rate of approx. 50 cm/h) approx. 23 mg KLH per ml gel werebound and even with the highest flow rate of 8.0 ml/min (correspondingto a linear flow rate of approx. 100 cm/h) the achieved binding ofapprox. 17 mg KLH per ml gel was excellent. Break through of KLH wasmonitored by UV detection at 280 and 340 nm. Subsequently further trialswere initiated in lab scale as well as during scale up to optimize thechromatography parameter due to an effective and economic isolation andpurification process.

In addition in situ dissociation was tested on KLH captured on thePoros® 50 HQ column applying the chromatography parameters as describedabove. After a first rinsing step with TRIS/HCl buffer (pH 7.4,including CaCl₂, MgCl₂ and NaCl) buffer exchange with glycine/NaOHbuffer (pH 9.6, including NaCl, EDTA) was performed. No breakthrough ofKLH was observed during rinsing the column under dissociationconditions.

Elution of the KLH subunit fraction was achieved using a elutiongradient of 0.075 to 0.575 M sodium chloride, glycine/NaOH buffer (pH9.6, including NaCl, EDTA).

The degree of dissociation was determined by means of analyticalMPLC/SEC (FPLC®; separating column: Superose® 6 HR 10/30, molecularweight separation range 5×103-5×106 Da) and by means of native PAGE. Themeasurement of the subunit fraction showed that an almost quantitativedissociation into the KLH subunits was achieved on the column. Due tothe concentration of KLH subunits (approx. 20 mg/ml) the filteredfraction (0.22 μm membrane filter) was used for further purification bygel filtration without any additional processing.

At the end of the research program, anion exchange chromatography wasthe method of choice.

1. An anion exchange chromatography column comprising a matrix materialwith a particle size of more than 30 μm and a pore size of between 3,000and 8,000 Angstroms.
 2. A method of isolating and/or purifyinghemocyanin or immunocyanin comprising the steps of: a) providing ahemocyanin formulation derived from a marine mollusk; b) reducing theconductivity of the formulation provided in a) to a value of between 30mS/cm and 10 mS/cm by adding a dilution buffer; c) adding the dilutedhemocyanin formulation obtained in step b) to an anion exchangechromatography column; d) rinsing the column with a buffer to purify thehemocyanin, preferably to remove salts, and other proteins; and e)eluting the hemocyanin or immunocyanin from the column by adding asecond buffer.
 3. The method according to claim 2 wherein the buffer forpurifying is a buffer in the pH between 7 and
 8. 4. The method of claim2, wherein hemocyanin is eluted, wherein a buffer of a pH between 7 and8 is used which includes Ca⁺⁺ and Mg⁺⁺ ions.
 5. The method of claim 2,wherein immunocyanin is eluted, wherein a buffer is employed of a pHbetween 9 and 10 which is free from Ca⁺⁺ and Mg⁺⁺ ions.
 6. The method ofclaim 2, wherein the matrix material is an anion exchange matrixincluding particles of a particle size of more than 30 nm, preferably 50μm+/−5 μm.
 7. The method of claim 2, wherein the anion exchange matrixincludes pores of a pore size between 3,000 and 8,000 Angstroms. 8.(canceled)
 9. The method of claim 3, wherein hemocyanin is eluted,wherein a buffer of a pH between 7 and 8 is used which includes Ca++ andMg++ ions.
 10. The method of claim 3, wherein immunocyanin is eluted,wherein a buffer of a pH between 7 and 8 is used which includes Ca++ andMg++ ions.
 11. The method of claim 4, wherein the matrix material is ananion exchange matrix including particles of a particle size of morethan 30 nm, preferably 50 μm+/−5 μm.
 12. The method of claim 5, whereinthe matrix material is an anion exchange matrix including particles of aparticle size of more than 30 nm, preferably 50 μm+/−5 μm.
 13. Themethod of claim 9, wherein the matrix material is an anion exchangematrix including particles of a particle size of more than 30 nm,preferably 50 μm+/−5 μm.
 14. The method of claim 10, wherein the matrixmaterial is an anion exchange matrix including particles of a particlesize of more than 30 nm, preferably 50 μm+/−5 μm.
 15. The method ofclaim 13, wherein the anion exchange matrix includes pores of a poresize between 3,000 and 8,000 Angstroms.
 16. The method of claim 14,wherein the anion exchange matrix includes pores of a pore size between3,000 and 8,000 Angstroms.
 17. The method of claim 3, wherein the anionexchange matrix includes pores of a pore size between 3,000 and 8,000Angstroms.
 18. The method of claim 4, wherein the anion exchange matrixincludes pores of a pore size between 3,000 and 8,000 Angstroms.
 19. Themethod of claim 5, wherein the anion exchange matrix includes pores of apore size between 3,000 and 8,000 Angstroms.